This application responds to NHLBI and the Office of Rare Diseases to support novel approaches to understand, prevent, and treat rare heart diseases. These exploratory projects must present new hypotheses and develop ways to demonstrate feasibility. Our goal is to understand why 3-10% of patients who have Fontan surgery to correct congenital univentricular hearts develop protein-losing enteropathy (PLE) months to years after the operation. Half of the PLE patients die. The variable onset suggests a multifactorial etiology. We hypothesize that genetic limitations and environmental insults lead to PLE, whose molecular basis is also unknown. Recent results show the PLE-stricken post-Fontan patients lose heparan sulfate proteoglycan (HSPG) specifically from the basolateral surface of small intestine epithelial cells. Our past work shows that some patients with inherited protein glycosylation disorders develop PLE and show an identical HSPG loss, which normalizes again when PLE resolves. This makes enterocyte HSPG the only known molecular marker of PLE in a relevant cell type. We hypothesize that localized loss of HSPG, plus a general systemic pro-inflammatory condition within the Fontan-induced setting of increased venous pressure cause PLE. Our preliminary in vitro data supports a robust synergism of these factors. In AIM 1, we will test this hypothesis in vitro by measuring paracellular protein leakage in human cultured epithelial cells variably stripped of their HSPG, challenged with cytokines, and placed under pressure. In AIM 2, mouse models that genetically lack the major basolateral HSPG core protein, Syndecan-1, or all HS synthesized by intestinal epithelial cells will be assessed for protein loss with an inflammatory challenge and/or increased pressure. We predict that the HSPG-deficient mice and intestinal mucosa derived from them will be more sensitive to inflammation-induced PLE. The results could provide a fundamental understanding of how PLE develops, identify Fontan patients genetically at risk for developing PLE, and provide insights to new therapeutics for this enigmatic rare disease.